Coronary heart disease and ischemic stroke are the main causes of morbidity and mortality in diabetic patients. Endothelial injury and dysfunction are common starting points for diabetic angiopathy. It is thus important to investigate the cellula and molecular mechanisms involved in coronary vascular endothelial dysfunction in diabetes. Endothelial function is regulated by changes in cytosolic Ca2+ concentration ([Ca2+]cyt) in endothelial cells (ECs). [Ca2+]cyt is controlled by the Ca2+ mobilization from intracellular stores coupled to Ca2+ influx from the external medium. In ECs, the endoplasmic reticulum (ER) accounts for approximately 75% of the total intracellular Ca2+ stores and the [Ca2+] in the ER ([Ca2+]ER) determines in great part the generation of important Ca2+ signals. The objective of this study is to examine whether [Ca2+]ER is altered in diabetic ECs and how abnormal [Ca2+]ER leads to vascular endothelial dysfunction in diabetic heart. In Aim 1, we will examine whether and how the rise in [Ca2+]cyt due to Ca2+ release from the ER (indicative of the level of [Ca2+]ER) and [Ca2+]ER are attenuated in coronary ECs isolated from diabetic mice. The ER membrane constitutes Ca2+ pumps (sarco/endoplasmic reticulum Ca2+ ATPase (SERCA)) and several classes of intracellular Ca2+ releasing channels. Recently, stromal interaction molecule (STIM) was identified as an essential protein for the store-operated Ca2+ entry (SOCE) and a contributor to the Ca2+ refilling into the ER by interacting with SERCA on the ER membrane. In Aim 2, we will investigate the potential role of STIM1 in decreased [Ca2+]ER in diabetic coronary ECs. Our preliminary data show that SERCA3 and STIM1 protein expressions are markedly decreased in diabetic coronary ECs and STIM1 overexpression in diabetic coronary ECs significantly increases Ca2+ leak from the ER. In addition, Ca2+ transported to mitochondria from the ER plays a crucial role in cell apoptosis; increased mitochondrial Ca2+ ([Ca2+]mit) can trigger mitochondrial fragmentation, loss of mitochondrial integrity and cell death in many cell types. Our results show that [Ca2+]mit is significantly increased in coronary ECs in diabetes. In Aim 3, we will define whether Ca2+ transportation from the ER to mitochondria is increased in coronary ECs in diabetes and examine the role of the Ca2+ communication between the ER and mitochondria in endothelial dysfunction in diabetic heart. To examine Ca2+ handling by the ER in diabetic coronary ECs will further our understanding of pathophysiological aspects of coronary vascular complications in diabetes. Completion of this study will provide important insights into developing new therapeutic interventions for cardiac ischemia in diabetes.